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Inhibition by opioids of phagocytosis in peritoneal macrophages
Neuropeptides (1991) 18, S-40 @ Longman Group UK Ltd 1991
Inhibition by Opioids Macrophages A, M. CASELLAS, H. GUARDIO~ BiologyDepartment,
of Phagocytosis
in Peritoneal
and F. L. RE~AUD
University of Puerto Rico, Rio Piedras, Puerto Rico 00937 (Reprint requests to FLR)
Abstract-We have tested the effect of prototypic opioid agonists on phagocytosis of sheep erythrocytes by mouse peritoneal macrophages. It was found that morphine and all the opioid peptides tested inhibited phagocytosis by a biphasic, naloxone-reversible mechanism. Delta agonists were the most effective inhibitors, suggesting that the response is mediated by a delta receptor. Chronic exposure to morphine apparently results in the development of tolerance since under these conditions the inhibitor effect of the opiate is abolished. These results are similar to previously reported effects of opioids on endocytosis in other systems, which suggests that this inhibition is part of a basic regulatory mechanism that has been conserved in evolution.
Introduction Opioids affect a wide variety of processes through receptors located in the central and peripheral nervous system. In general, they seem to inhibit impulse transmission and neurotransmitter discharge by altering membrane permeability to cations and by inhibiting adenylate cyclase (1). However, recent evidence demonstrates that opioids can also have a number of effects on cells of the immune system, such as enhancing the cytotoxicity of peripheral blood lymphocytes and stimulating monocyte chemotaxis (2, 3). These suggest that opioid receptors are not restricted in their dist~bution to the nervous system and can Date received 12 April 1990 Date accepted 18 July 1990
also be found in non-nervous tissues. Furthermore, these findings substantiate the hypothesis that there is a great deal of interaction between the nervous and the immune system, and that chemical signals secreted by cells of the nervous system can modulate the function of immunocytes (3). In previous work it was demonstrated that met-enkephalin inhibited phagocytosis in mouse peritoneal macrophages and it was postulated that this effect was mediated by a delta-like receptor (4, 5). We now confirm and extend these observations and provide further evidence for the role of a delta-like receptor in the inhibition by opioids of phagocytosis in mouse peritoneal macrophages. In addition we demonstrate that chronic exposure of macrophages to morphine apparently results in tolerance to the inhibitory effects of the drug.
36 Materialsand Methods Ceils Thioglycollate elicited macrophages (6) were obtained from C3HeB/FeJ female mice 6-8 weeks old (Jackson Labs, Bar Harbor, ME) in the following manner: each mouse was injected i.p. with 3ml of Brewer’s Thioglycollate medium (Difco Detroit, MI). The mice were killed after 5 days by cervical dislocation and the peritoneal cavity was washed with lOm1 of PBS. After a 2 min massage the cell exudate was removed with a syringe, centrifuged for 10 min at 250g and 4°C. The cell sediment was then resuspended for 30s in cold 0.2% NaCl and immediately mixed with an equal volume of cold 1.6% NaCl in order to lyse residual RBC that may be present. The cells were then washed with Hank’s balanced saline (HBSS) by centrifugation, counted using an electronic cell counter (Coulter Electronics, Hialeah, FLA) and the number of viable cells (over 90%) determined by Trypan Blue exclusion. Macrophages were plated in eight-well Lab-Tek chambers (Miles Laboratories, Naperville, IL) at a concentration of 2.5 x lo5 cells per ml, 0.35ml per well, at 37°C in 5% coa. Sheep erythrocytes (SRBC, Colorado Serum, CO) were opsonized with a l/3000-dilution of IgG anti-SRBC (Diamedic Cordis Labs) in the following way: SRBC were washed with HBSS by centrifugation, the antibody was added, and the mixture was incubated at 30°C for 1.5 min with gentle agitation; this was followed by three washes with HBSS by centrifugation. Phagocy tosis assay
Macrophage cultures were washed with HBSS followed by the addition of either RPM1 1640 medium (GIBCO Labs, Grand Island, NY) or opioid agonist in RPMI. After a 30 min incubation opsonized sheep red blood cells (SRBC) were added to the macrophages at a ratio of 160 RBC per macrophage and incubated for 30 min at 37°C in 5% COz. In some experiments the incubation time prior to addition of SRBC was varied to determine the optimal assay conditions. The effect of a chronic exposure to morphine on phagocytosis was also determined by culturing macrophages overnight (approximately 16h) in the presence of
NEUROPEFTIDES
50 nM morphine prior to the phagocytosis assay. In all experiments unopsonized SRBC were added to some macrophage samples as controls. Phagocytosis was stopped by placing the cells on ice, and non-ingested SRBC were then lysed by hypotonic shock (7). After fixation in 1% glutaraldehyde and staining with Giemsa, phagocytosis was measured by light microscopy. The results were expressed in terms of percent phagocytosis (percentage of cells ingesting at least one SRBC) or in terms of the phagocytosis factor: percent phagocytosis of cells in the presence of agonis~percent phagocytosis of control cells x 100. At least 100 macrophages were scored per well and each experiment was performed at least 3 times. Each figure illustrates the results of a representative experiment and the values displayed in each figure represent the mean and standard deviation of triplicate replicas. The significance of any difference observed was determined by means of the Students’ t-test or by the analysis of variance. Redts The ingestion of SRBC by macrophages at different time intervals and the inhibitory effect of morphine on this process is shown in Figure 1. The ingestion of cells reaches a plateau at around 20 min and the drug reduces the level of phagocytosis at the plateau by about 20% (P < 0.01). Increasing the pre-incubation time with morphine to 1 h had no significant effect on the results obtained. Therefore, all the subsequent experiments were done with a pre-incubation time with the agonist of 30 min and an incubation time with the SRBC of 30 min. The dose-response curve of met- and leu-enkephalin and their amides on phagocytosis is illustrated in Figure 2. All these peptides appear to be similarly effective in inhibiting phagocytosis (PF = 0.61-0.68) in a wide concentration range, between 1 nM and 500nM; at these concentrations the differences when compared to control cells were significant according to variance analysis (P < 0.05). However, leu-enkephalinamide appears to be the most potent compound, with a maximal inhibitory concentration of 10nM. Similar results were obtained with mu agonists (Fig. 3A) although morphine appeared to be less
37
INHIBITION BY OPIOIDS OF PHAGOCYTOSIS IN PERITONEAL MACROPHAGES
io
lo
TIME (M;oN) Fig. 1 Phagocytosis of SRBC by mouse peritoneal macrophages in the absence (A) and presence (A) of 50nM morphine.
effective (PF = 0.80 rt 0.04). Both dyno~hin (1-13) and p-endorphin appear to be far less effective than the enkephalins (Fig. 3B); however, the effect’ of both these agonists and the mu agonists was significant when compared to control cells in a concentration range between 1nM and 500nM, according to variance analysis. The data on the potency and efficacy of the different agonists tested are summa~zed in the Table. The inhibitory effect of met- and leu-enkephalin on macrophage phagocytosis was naloxone-reversible (Fig. 4) and similar results were obtained for all the other opioids tested (data not shown). When macrophages were chronically exposed to morphine the cells apparently developed tolerance to the drug, since they were no longer inhibited by it (Fig. 5). However, withdrawal of morphine from the putatively tolerant cells had no significant effect on the basal rate of phagocytosis.
mediated by a delta-type receptor. We have extended those obse~ations and have found that other delta agonists, such as met-enkephalinamide, leu-enkephalin and leu-enkephalinamide, also inhibit this process in a similar way. It is of interest to note that met-enkephalinamide appears to be the most potent agonist tested, since it has its maximal effect at a concentration of 10nM. The mu agonists tested, morphine and morphiceptin, were slightly less effective, whereas both p-endorphin (E) and dynorphin (K) were much weaker inhibitors. This strengthens the hypothesis that this effect is mediated by a delta-type receptor. Further support comes from a recent report that a delta specific ligand labels a 58Kd band in a macrophage cell line, P388dr (8); similar proteins have been reported in B and T cells (9). A kappa-like binding site was also reported to be present in P388di cells using a kappa specific ligand; however this site should be explored further because dyno~hin (l-13) was not able to displace the ligand except at micromolar concentrations (8). In our work we found that dynorphin
Discussion Our results confirm previous studies which reported that met-enkephalin inhibited phagocytosis in mouse peritoneal macrophages in a concentration range of 1 through 100 nM through a naloxone-reversible mechanism (4). These researchers postulated that the inhibition of phagocytosis by met-enkephalin in macrophages was
+ 1
1
a*
log
1000
Cont. &Y
Fig. 2 Dose-response curves of delta agonists on phagocytosis of SRBC by mouse peritoneal macrophages: panel A, leuenkephalin (Ci), leu-enkeph~inamide (e); panel B, met-enkephaiin (!I), met-enkephahnamide (a). The dashed line indicates the control level of phagocytosis.
38
NEUROPEPTIDES
. I
.
il#Cont.
1000
(n!7
Fig. 3 Dose-response curves of mu, kappa and epsilon agonists on phagocytosis of SRBC by mouse peritoneal macrophages: panel A, morphine (0). morphiceptin (4); panel B, B-endorphin (Cl), dynorphin 1-13 (+). The dashed line indicates the control level of phagocytosis.
(1-13) had only weak effects on phagocytosis; therefore, if a kappa receptor exists in macrophages it does not seem to be mechanistically coupled to phagocytosis. Petty and Berg (10) have reported that metenkephalin at 10 nM stimulates phagocytosis in the murine macrophage cell line RAW264 and in mouse peritoneal macrophages. However, we
Table
Comparison
of the
inhibitory
potencies
believe that these apparently contradictory results are due to fundamental differences in procedure. Foris et al (4, 5), who made the original report of the inhibitory effect of met-enkephalin on phagocytosis, also reported a stimulation of this process by the opioid peptide at the higher agonist concentrations (around 1uM); however the effects of met-enkephalin at this higher concentration were not naloxone-reversible, suggesting that they were not mediated by an opioid receptor. Furthermore, other effects of met-enkephalin at high concentrations were not observed in the presence of an enkephalinase inhibitor. Thus, it could be assumed that the prolonged incubation in the presence of SRBC (3 h) that was utilized in the work of Petty and Berg (10) resulted in the degradation of met-enkephalin, and one of the degradation products might be responsible for the stimulatory effect observed. Therefore, it would be of interest to know if the results of Petty and Berg (10) are naloxone-reversible and preventable by an enkephalinase inhibitor. Morphine has also been reported to inhibit phagocytosis in peritoneal macrophages obtained from mice treated for several days with increasing concentrations of the drug (11). These mice were found to be clearly more susceptible to infections than control, untreated mice and the authors speculated that this could probably explain at least in part the susceptibility to infections observed in drug addicts. This susceptibility is usually explained as due to the use and sharing of dirty needles and this problem is probably exacerbated
of opioid
agonists
on
phagocytosis
by mouse
peritoneal
macrophages
Receptor subtype
delta
mu kappa epsilon
Agonist
leu-enkephalinamide met-enkephalinamide leu-enkephalin met-enkephalin morphine morphiceptin dynorphin (1-13) B-endorphin
’ Expressed in terms of the phagocytosis factor +SD.
Apparent optimal inhibitory concentration (nM)
10 50 500 100 50 100 50 500
Maximal inhibition’
0.64 0.68 0.66 0.61 0.80 0.71 0.83 0.82
+ + f + f + + +
0.08 0.07 0.02 0.02 0.04 0.07 0.06 0.04
INHIBITION BY OPIOIDS OF PWAGOCYTOSIS
C
IN PERITONEAL
1-E
L-E
+NX
4 Reversion by naloxone of the inhibition by delta agonists of phagocytosis by mouse peritoneal macrophages. Open bar, control cells (C) phagocytising in the absence of agonist; hatched bars, cells phagocytising in the presence of 1OOnMmet-enkephahn and in the absence (M-E), or presence of 1~nM naloxone (M-E + NX); black bars, cells phagocytising in the presence of 5OOnM leu-enkephalin and in the absence (L-E) or presence of SO&M naloxone (L-E + NX). The asterisk indicates a value that is significantly different from that of control: cells (P < 0.01). Fig.
by the effect of the opioid on the immune defenses. However, in our present work we demonstrate that a chronic exposure of macrophages to morphine in vitro apparently results in the development of tolerance to the inhibitory effect of the opioid. It would be of interest to determine if the intermittent use of drugs by addicts also results in the development of tolerance to these effects. The potency of agonists with respect to a receptor is conventionally expressed in terms of the EC& However, both in other reports (3-5) and in the present paper the dose-response curves obtained were biphasic and U-shaped. A determination of EC30 values from such curves would be of questionable value, since these curves are most likely the result of two antagonic processes taking place simultaneously. Similarly shaped curves have been reported previously in our laboratory for the inhibition by opioids of phagocytosis in the protozoan ciliate Tetrahymena (12) and by other workers for the inhibition by opioids of pinocytosis in Amoeba (13). We have speculated that the decreased response to the agonist at higher concentrations could be due to either a desensitization of the receptor or to the activation of a stimulatory set of receptors; for example, biphasic curves are
39
MACROPHAGES
obtained as a result of the desensitization of the acetylcholine receptor by high ~ncentrations of carbamylcholine (14). Another similarity between the effect of opioids on phagocytosis in macrophages and in Tetrahymena is that, as we have reported recently, a chronic exposure to morphine also results in the apparent development of tolerance in this protozoan ciliate (15). Therefore, it is interesting that opioids appear to modulate in a similar fashion endocytic processes in macrophages and protozoa, suggesting that the mechanism involved may have appeared early in evolution and has been conserved. However, in the case of Te~ruhymenuwithdrawal of the agonist results in a significant decrease in the basal rate of phagocytosis, which suggests that a process akin to dependence has developed in this organism. This is in contrast to the data presented in this work that shows that the basal rate of phagocytosis of tolerant macrophages is not affected by withdrawal of mo~hine from the medium. The molecular basis of tolerance still needs to be established in both Tetrahymena and macrophages; in other systems it seems to involve changes in the transduction machinery (16). Opioids are known to have other effects on
“I
s
0.1) i
Y--
! M
M
A
Fig. 5 Effect of acute and chronic exposures to morphine on phagocytosis of SRBC by mouse peritoneal macrophages. Open bars: C, control cells phagocytising in the absence of morphine; M, in the presence of 5OnM morphine after a 30 min pre-incubation with the drug. Hatched bars: cells cultured for 16 h in the presence of 50nM morphine and phagocytising in the presence (M) and the absence (A) of the drug. The asterisk indicates a value that is significantly different from that of control cells (P < 0,Ol).
40
NEUROPEPTIDES
immunocytes, such as the stimulation of chemotaxis and of cytotoxicity (2, 3). These processes were not studied in the present work, and therefore it is not known how they would be affected by the various opioid agonists tested or by the development of tolerance. Nevertheless, our findings contribute to substantiate further the existence of strong links between the nervous and the immune system. It could be suggested, for example, that the secretion of opioids under stress could result in immunosuppression due to the inhibition of the phagocytic cells of the immune system. Further work should shed light on how these neuromodulators affect immune functions, both in control organisms and in tolerant ones.
7.
8.
9.
Acknowledgements We wish to express our appreciation to Dr Ivette GarciaCastro, from the Biology Department, University of Puerto Rico, Rio Piedras Campus, for her help with macrophage techniques. This work was supported by NIH Grant GM08102, the MARC Honors Program and the FIPI Program of the University of Puerto Rico, Rio Piedras Campus.
10.
11.
References IL.
North, R. A. (1986) Membrane conductances and opioid receptor subtypes. In: Brown, R. M., Clouet, D. H. & Friedman, D. P. (eds.) NIDA Research Monographs, Series 71. U.S. Dept. of Health and Human Services. Carr, D. J. J. and Blalock, J. E. (1986) A molecular basis for bidirectional communication between the immune and neuroendocrine systems. In: Cinader, B. and Miller, R. G. (eds) Progress in Immunology. Academic Press, Orlando, p. 619-628. Ruff, M. R. and Pert, C. B. (1986) Neuropeptides as chemoattractants for human monocytes and tumor cells: a basis for mind-body communication. In: Plotnikoff, M. P., Faith, R. E., Murgo, A. J. and Good, R. A. (eds) Enkephalins and endorphins, stress and the immune system. Plenum Publishing Corp., p. 387-398.
13.
14.
15.
16.
Foris, G.. Medgyesi, G. A., Gyimesi, E. and Hauck, M. (1984) Met-enkephalin induced alterations of macrophage functions. Mol. Immunol. 21: 747-750. Foris, G., Medgyesi, G. A. and Hauck, M. (1986) Bidirectional effect of met-enkephalin on macrophage effector functions. Mol. Cell. Biochem. 76: 127-137. Herscowitz, H. B., Holden, H. T., Bellanti. J. A. and Ghaffar, A. (eds) (1981) Manual of macrophage methodology. Collection, chracterization, and function. Marcel Dekker, Inc., New York. Bobak, D. A., Gaither, T. A., Frank, M. M. and Tenner, A. J. (1987) Modulation of FcR function by complement: subcomponent Clq enhances the phagocytosis of IgGopsonized targets by human monocytes and culturederived macrophages. J. Immunol. 138: 1150-1156. Carr. D. J. J., DeCosta. B. R., Kim, C.-H., Jacobson, A. E., Guarcello, V., Rice, K. C. and Blalock, J. E. (1989) Opioid receptors on cells of the immune system: evidence for 6- and K-classes. J. Endocrinol. 122: 161-168. Carr, D. J. J., Kim, C-H.. DeCosta, B., Jacobson, A. E., Rice, K. C. annd Blalock, J. E. (1988) Evidence for a &class opioid receptor on cells of the immune system. Cell. Immunol. 116: 44-51. Petty, H. R. and Berg, K. A. (1988) Combinative ligandreceptor interactions: epinephrine depresses RAW264 macrophage antibody-dependent phagocytosis in the absence and presence of met-enkephalin. J. Cell. Physiol. 134: 281-286. Tubaro, E., Borelli, G., Croce, C., Cavallo, G. and Santiangeli, C. (1983) Effect of morphine on resistance to infection. J. Infect. Dis. 148: 656-666. De Jesus, S. and Renaud, F. L. (1989) Phagocytosis in Tetrahymena thermophila: naloxone-reversible inhibition by opiates. Comp. Biochem. Physiol. 92C: 139-142. Josefsson, J. 0. and Johansson, P. (1979) Naloxone-reversible effect of opioids on pinocytosis in Amoeba proteus. Nature 282: 78-80. Boksa, P. and Livett, B. G. (1984) Desensitization to nicotinic cholinergic agonists and K’ agents that stimulate catecholamine secretion in isolated adrenal chromaffin cells. J. Neurochem. 42: 607-617. Salaman, A., Roman, M., Renaud, F. L. and Silva, W. I. (1990) Effect of chronic opioid treatment on phagocytosis in Tetrahymena. Neuropeptides (in press). Koob, G. F. and Bloom, F. E. (1988) Cellular and molecular mechanisms of drug dependence. Science 242: 715-723.
Inhibition by Opioids Macrophages A, M. CASELLAS, H. GUARDIO~ BiologyDepartment,
of Phagocytosis
in Peritoneal
and F. L. RE~AUD
University of Puerto Rico, Rio Piedras, Puerto Rico 00937 (Reprint requests to FLR)
Abstract-We have tested the effect of prototypic opioid agonists on phagocytosis of sheep erythrocytes by mouse peritoneal macrophages. It was found that morphine and all the opioid peptides tested inhibited phagocytosis by a biphasic, naloxone-reversible mechanism. Delta agonists were the most effective inhibitors, suggesting that the response is mediated by a delta receptor. Chronic exposure to morphine apparently results in the development of tolerance since under these conditions the inhibitor effect of the opiate is abolished. These results are similar to previously reported effects of opioids on endocytosis in other systems, which suggests that this inhibition is part of a basic regulatory mechanism that has been conserved in evolution.
Introduction Opioids affect a wide variety of processes through receptors located in the central and peripheral nervous system. In general, they seem to inhibit impulse transmission and neurotransmitter discharge by altering membrane permeability to cations and by inhibiting adenylate cyclase (1). However, recent evidence demonstrates that opioids can also have a number of effects on cells of the immune system, such as enhancing the cytotoxicity of peripheral blood lymphocytes and stimulating monocyte chemotaxis (2, 3). These suggest that opioid receptors are not restricted in their dist~bution to the nervous system and can Date received 12 April 1990 Date accepted 18 July 1990
also be found in non-nervous tissues. Furthermore, these findings substantiate the hypothesis that there is a great deal of interaction between the nervous and the immune system, and that chemical signals secreted by cells of the nervous system can modulate the function of immunocytes (3). In previous work it was demonstrated that met-enkephalin inhibited phagocytosis in mouse peritoneal macrophages and it was postulated that this effect was mediated by a delta-like receptor (4, 5). We now confirm and extend these observations and provide further evidence for the role of a delta-like receptor in the inhibition by opioids of phagocytosis in mouse peritoneal macrophages. In addition we demonstrate that chronic exposure of macrophages to morphine apparently results in tolerance to the inhibitory effects of the drug.
36 Materialsand Methods Ceils Thioglycollate elicited macrophages (6) were obtained from C3HeB/FeJ female mice 6-8 weeks old (Jackson Labs, Bar Harbor, ME) in the following manner: each mouse was injected i.p. with 3ml of Brewer’s Thioglycollate medium (Difco Detroit, MI). The mice were killed after 5 days by cervical dislocation and the peritoneal cavity was washed with lOm1 of PBS. After a 2 min massage the cell exudate was removed with a syringe, centrifuged for 10 min at 250g and 4°C. The cell sediment was then resuspended for 30s in cold 0.2% NaCl and immediately mixed with an equal volume of cold 1.6% NaCl in order to lyse residual RBC that may be present. The cells were then washed with Hank’s balanced saline (HBSS) by centrifugation, counted using an electronic cell counter (Coulter Electronics, Hialeah, FLA) and the number of viable cells (over 90%) determined by Trypan Blue exclusion. Macrophages were plated in eight-well Lab-Tek chambers (Miles Laboratories, Naperville, IL) at a concentration of 2.5 x lo5 cells per ml, 0.35ml per well, at 37°C in 5% coa. Sheep erythrocytes (SRBC, Colorado Serum, CO) were opsonized with a l/3000-dilution of IgG anti-SRBC (Diamedic Cordis Labs) in the following way: SRBC were washed with HBSS by centrifugation, the antibody was added, and the mixture was incubated at 30°C for 1.5 min with gentle agitation; this was followed by three washes with HBSS by centrifugation. Phagocy tosis assay
Macrophage cultures were washed with HBSS followed by the addition of either RPM1 1640 medium (GIBCO Labs, Grand Island, NY) or opioid agonist in RPMI. After a 30 min incubation opsonized sheep red blood cells (SRBC) were added to the macrophages at a ratio of 160 RBC per macrophage and incubated for 30 min at 37°C in 5% COz. In some experiments the incubation time prior to addition of SRBC was varied to determine the optimal assay conditions. The effect of a chronic exposure to morphine on phagocytosis was also determined by culturing macrophages overnight (approximately 16h) in the presence of
NEUROPEFTIDES
50 nM morphine prior to the phagocytosis assay. In all experiments unopsonized SRBC were added to some macrophage samples as controls. Phagocytosis was stopped by placing the cells on ice, and non-ingested SRBC were then lysed by hypotonic shock (7). After fixation in 1% glutaraldehyde and staining with Giemsa, phagocytosis was measured by light microscopy. The results were expressed in terms of percent phagocytosis (percentage of cells ingesting at least one SRBC) or in terms of the phagocytosis factor: percent phagocytosis of cells in the presence of agonis~percent phagocytosis of control cells x 100. At least 100 macrophages were scored per well and each experiment was performed at least 3 times. Each figure illustrates the results of a representative experiment and the values displayed in each figure represent the mean and standard deviation of triplicate replicas. The significance of any difference observed was determined by means of the Students’ t-test or by the analysis of variance. Redts The ingestion of SRBC by macrophages at different time intervals and the inhibitory effect of morphine on this process is shown in Figure 1. The ingestion of cells reaches a plateau at around 20 min and the drug reduces the level of phagocytosis at the plateau by about 20% (P < 0.01). Increasing the pre-incubation time with morphine to 1 h had no significant effect on the results obtained. Therefore, all the subsequent experiments were done with a pre-incubation time with the agonist of 30 min and an incubation time with the SRBC of 30 min. The dose-response curve of met- and leu-enkephalin and their amides on phagocytosis is illustrated in Figure 2. All these peptides appear to be similarly effective in inhibiting phagocytosis (PF = 0.61-0.68) in a wide concentration range, between 1 nM and 500nM; at these concentrations the differences when compared to control cells were significant according to variance analysis (P < 0.05). However, leu-enkephalinamide appears to be the most potent compound, with a maximal inhibitory concentration of 10nM. Similar results were obtained with mu agonists (Fig. 3A) although morphine appeared to be less
37
INHIBITION BY OPIOIDS OF PHAGOCYTOSIS IN PERITONEAL MACROPHAGES
io
lo
TIME (M;oN) Fig. 1 Phagocytosis of SRBC by mouse peritoneal macrophages in the absence (A) and presence (A) of 50nM morphine.
effective (PF = 0.80 rt 0.04). Both dyno~hin (1-13) and p-endorphin appear to be far less effective than the enkephalins (Fig. 3B); however, the effect’ of both these agonists and the mu agonists was significant when compared to control cells in a concentration range between 1nM and 500nM, according to variance analysis. The data on the potency and efficacy of the different agonists tested are summa~zed in the Table. The inhibitory effect of met- and leu-enkephalin on macrophage phagocytosis was naloxone-reversible (Fig. 4) and similar results were obtained for all the other opioids tested (data not shown). When macrophages were chronically exposed to morphine the cells apparently developed tolerance to the drug, since they were no longer inhibited by it (Fig. 5). However, withdrawal of morphine from the putatively tolerant cells had no significant effect on the basal rate of phagocytosis.
mediated by a delta-type receptor. We have extended those obse~ations and have found that other delta agonists, such as met-enkephalinamide, leu-enkephalin and leu-enkephalinamide, also inhibit this process in a similar way. It is of interest to note that met-enkephalinamide appears to be the most potent agonist tested, since it has its maximal effect at a concentration of 10nM. The mu agonists tested, morphine and morphiceptin, were slightly less effective, whereas both p-endorphin (E) and dynorphin (K) were much weaker inhibitors. This strengthens the hypothesis that this effect is mediated by a delta-type receptor. Further support comes from a recent report that a delta specific ligand labels a 58Kd band in a macrophage cell line, P388dr (8); similar proteins have been reported in B and T cells (9). A kappa-like binding site was also reported to be present in P388di cells using a kappa specific ligand; however this site should be explored further because dyno~hin (l-13) was not able to displace the ligand except at micromolar concentrations (8). In our work we found that dynorphin
Discussion Our results confirm previous studies which reported that met-enkephalin inhibited phagocytosis in mouse peritoneal macrophages in a concentration range of 1 through 100 nM through a naloxone-reversible mechanism (4). These researchers postulated that the inhibition of phagocytosis by met-enkephalin in macrophages was
+ 1
1
a*
log
1000
Cont. &Y
Fig. 2 Dose-response curves of delta agonists on phagocytosis of SRBC by mouse peritoneal macrophages: panel A, leuenkephalin (Ci), leu-enkeph~inamide (e); panel B, met-enkephaiin (!I), met-enkephahnamide (a). The dashed line indicates the control level of phagocytosis.
38
NEUROPEPTIDES
. I
.
il#Cont.
1000
(n!7
Fig. 3 Dose-response curves of mu, kappa and epsilon agonists on phagocytosis of SRBC by mouse peritoneal macrophages: panel A, morphine (0). morphiceptin (4); panel B, B-endorphin (Cl), dynorphin 1-13 (+). The dashed line indicates the control level of phagocytosis.
(1-13) had only weak effects on phagocytosis; therefore, if a kappa receptor exists in macrophages it does not seem to be mechanistically coupled to phagocytosis. Petty and Berg (10) have reported that metenkephalin at 10 nM stimulates phagocytosis in the murine macrophage cell line RAW264 and in mouse peritoneal macrophages. However, we
Table
Comparison
of the
inhibitory
potencies
believe that these apparently contradictory results are due to fundamental differences in procedure. Foris et al (4, 5), who made the original report of the inhibitory effect of met-enkephalin on phagocytosis, also reported a stimulation of this process by the opioid peptide at the higher agonist concentrations (around 1uM); however the effects of met-enkephalin at this higher concentration were not naloxone-reversible, suggesting that they were not mediated by an opioid receptor. Furthermore, other effects of met-enkephalin at high concentrations were not observed in the presence of an enkephalinase inhibitor. Thus, it could be assumed that the prolonged incubation in the presence of SRBC (3 h) that was utilized in the work of Petty and Berg (10) resulted in the degradation of met-enkephalin, and one of the degradation products might be responsible for the stimulatory effect observed. Therefore, it would be of interest to know if the results of Petty and Berg (10) are naloxone-reversible and preventable by an enkephalinase inhibitor. Morphine has also been reported to inhibit phagocytosis in peritoneal macrophages obtained from mice treated for several days with increasing concentrations of the drug (11). These mice were found to be clearly more susceptible to infections than control, untreated mice and the authors speculated that this could probably explain at least in part the susceptibility to infections observed in drug addicts. This susceptibility is usually explained as due to the use and sharing of dirty needles and this problem is probably exacerbated
of opioid
agonists
on
phagocytosis
by mouse
peritoneal
macrophages
Receptor subtype
delta
mu kappa epsilon
Agonist
leu-enkephalinamide met-enkephalinamide leu-enkephalin met-enkephalin morphine morphiceptin dynorphin (1-13) B-endorphin
’ Expressed in terms of the phagocytosis factor +SD.
Apparent optimal inhibitory concentration (nM)
10 50 500 100 50 100 50 500
Maximal inhibition’
0.64 0.68 0.66 0.61 0.80 0.71 0.83 0.82
+ + f + f + + +
0.08 0.07 0.02 0.02 0.04 0.07 0.06 0.04
INHIBITION BY OPIOIDS OF PWAGOCYTOSIS
C
IN PERITONEAL
1-E
L-E
+NX
4 Reversion by naloxone of the inhibition by delta agonists of phagocytosis by mouse peritoneal macrophages. Open bar, control cells (C) phagocytising in the absence of agonist; hatched bars, cells phagocytising in the presence of 1OOnMmet-enkephahn and in the absence (M-E), or presence of 1~nM naloxone (M-E + NX); black bars, cells phagocytising in the presence of 5OOnM leu-enkephalin and in the absence (L-E) or presence of SO&M naloxone (L-E + NX). The asterisk indicates a value that is significantly different from that of control: cells (P < 0.01). Fig.
by the effect of the opioid on the immune defenses. However, in our present work we demonstrate that a chronic exposure of macrophages to morphine in vitro apparently results in the development of tolerance to the inhibitory effect of the opioid. It would be of interest to determine if the intermittent use of drugs by addicts also results in the development of tolerance to these effects. The potency of agonists with respect to a receptor is conventionally expressed in terms of the EC& However, both in other reports (3-5) and in the present paper the dose-response curves obtained were biphasic and U-shaped. A determination of EC30 values from such curves would be of questionable value, since these curves are most likely the result of two antagonic processes taking place simultaneously. Similarly shaped curves have been reported previously in our laboratory for the inhibition by opioids of phagocytosis in the protozoan ciliate Tetrahymena (12) and by other workers for the inhibition by opioids of pinocytosis in Amoeba (13). We have speculated that the decreased response to the agonist at higher concentrations could be due to either a desensitization of the receptor or to the activation of a stimulatory set of receptors; for example, biphasic curves are
39
MACROPHAGES
obtained as a result of the desensitization of the acetylcholine receptor by high ~ncentrations of carbamylcholine (14). Another similarity between the effect of opioids on phagocytosis in macrophages and in Tetrahymena is that, as we have reported recently, a chronic exposure to morphine also results in the apparent development of tolerance in this protozoan ciliate (15). Therefore, it is interesting that opioids appear to modulate in a similar fashion endocytic processes in macrophages and protozoa, suggesting that the mechanism involved may have appeared early in evolution and has been conserved. However, in the case of Te~ruhymenuwithdrawal of the agonist results in a significant decrease in the basal rate of phagocytosis, which suggests that a process akin to dependence has developed in this organism. This is in contrast to the data presented in this work that shows that the basal rate of phagocytosis of tolerant macrophages is not affected by withdrawal of mo~hine from the medium. The molecular basis of tolerance still needs to be established in both Tetrahymena and macrophages; in other systems it seems to involve changes in the transduction machinery (16). Opioids are known to have other effects on
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! M
M
A
Fig. 5 Effect of acute and chronic exposures to morphine on phagocytosis of SRBC by mouse peritoneal macrophages. Open bars: C, control cells phagocytising in the absence of morphine; M, in the presence of 5OnM morphine after a 30 min pre-incubation with the drug. Hatched bars: cells cultured for 16 h in the presence of 50nM morphine and phagocytising in the presence (M) and the absence (A) of the drug. The asterisk indicates a value that is significantly different from that of control cells (P < 0,Ol).
40
NEUROPEPTIDES
immunocytes, such as the stimulation of chemotaxis and of cytotoxicity (2, 3). These processes were not studied in the present work, and therefore it is not known how they would be affected by the various opioid agonists tested or by the development of tolerance. Nevertheless, our findings contribute to substantiate further the existence of strong links between the nervous and the immune system. It could be suggested, for example, that the secretion of opioids under stress could result in immunosuppression due to the inhibition of the phagocytic cells of the immune system. Further work should shed light on how these neuromodulators affect immune functions, both in control organisms and in tolerant ones.
7.
8.
9.
Acknowledgements We wish to express our appreciation to Dr Ivette GarciaCastro, from the Biology Department, University of Puerto Rico, Rio Piedras Campus, for her help with macrophage techniques. This work was supported by NIH Grant GM08102, the MARC Honors Program and the FIPI Program of the University of Puerto Rico, Rio Piedras Campus.
10.
11.
References IL.
North, R. A. (1986) Membrane conductances and opioid receptor subtypes. In: Brown, R. M., Clouet, D. H. & Friedman, D. P. (eds.) NIDA Research Monographs, Series 71. U.S. Dept. of Health and Human Services. Carr, D. J. J. and Blalock, J. E. (1986) A molecular basis for bidirectional communication between the immune and neuroendocrine systems. In: Cinader, B. and Miller, R. G. (eds) Progress in Immunology. Academic Press, Orlando, p. 619-628. Ruff, M. R. and Pert, C. B. (1986) Neuropeptides as chemoattractants for human monocytes and tumor cells: a basis for mind-body communication. In: Plotnikoff, M. P., Faith, R. E., Murgo, A. J. and Good, R. A. (eds) Enkephalins and endorphins, stress and the immune system. Plenum Publishing Corp., p. 387-398.
13.
14.
15.
16.
Foris, G.. Medgyesi, G. A., Gyimesi, E. and Hauck, M. (1984) Met-enkephalin induced alterations of macrophage functions. Mol. Immunol. 21: 747-750. Foris, G., Medgyesi, G. A. and Hauck, M. (1986) Bidirectional effect of met-enkephalin on macrophage effector functions. Mol. Cell. Biochem. 76: 127-137. Herscowitz, H. B., Holden, H. T., Bellanti. J. A. and Ghaffar, A. (eds) (1981) Manual of macrophage methodology. Collection, chracterization, and function. Marcel Dekker, Inc., New York. Bobak, D. A., Gaither, T. A., Frank, M. M. and Tenner, A. J. (1987) Modulation of FcR function by complement: subcomponent Clq enhances the phagocytosis of IgGopsonized targets by human monocytes and culturederived macrophages. J. Immunol. 138: 1150-1156. Carr. D. J. J., DeCosta. B. R., Kim, C.-H., Jacobson, A. E., Guarcello, V., Rice, K. C. and Blalock, J. E. (1989) Opioid receptors on cells of the immune system: evidence for 6- and K-classes. J. Endocrinol. 122: 161-168. Carr, D. J. J., Kim, C-H.. DeCosta, B., Jacobson, A. E., Rice, K. C. annd Blalock, J. E. (1988) Evidence for a &class opioid receptor on cells of the immune system. Cell. Immunol. 116: 44-51. Petty, H. R. and Berg, K. A. (1988) Combinative ligandreceptor interactions: epinephrine depresses RAW264 macrophage antibody-dependent phagocytosis in the absence and presence of met-enkephalin. J. Cell. Physiol. 134: 281-286. Tubaro, E., Borelli, G., Croce, C., Cavallo, G. and Santiangeli, C. (1983) Effect of morphine on resistance to infection. J. Infect. Dis. 148: 656-666. De Jesus, S. and Renaud, F. L. (1989) Phagocytosis in Tetrahymena thermophila: naloxone-reversible inhibition by opiates. Comp. Biochem. Physiol. 92C: 139-142. Josefsson, J. 0. and Johansson, P. (1979) Naloxone-reversible effect of opioids on pinocytosis in Amoeba proteus. Nature 282: 78-80. Boksa, P. and Livett, B. G. (1984) Desensitization to nicotinic cholinergic agonists and K’ agents that stimulate catecholamine secretion in isolated adrenal chromaffin cells. J. Neurochem. 42: 607-617. Salaman, A., Roman, M., Renaud, F. L. and Silva, W. I. (1990) Effect of chronic opioid treatment on phagocytosis in Tetrahymena. Neuropeptides (in press). Koob, G. F. and Bloom, F. E. (1988) Cellular and molecular mechanisms of drug dependence. Science 242: 715-723.